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User Bulletin
Applied Biosystems 3730/3730xl DNA Analyzers and
3130/3130xl Genetic Analyzers
July 2005
SUBJECT:
Using the SNaPshot® Multiplex System with the POP-7™
Polymer on Applied Biosystems 3730/3730xl DNA
Analyzers and 3130/3130xl Genetic Analyzers
In This User
Bulletin
This user bulletin includes the following topics:
Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
The SNaPshot Multiplex System with
POP-7 Polymer Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Protocol Validation and Testing. . . . . . . . . . . . . . . . . . . . . . . . . 2
Customer Acknowledgement. . . . . . . . . . . . . . . . . . . . . . . . . . . 2
SNaPshot Multiplexing on the Applied Biosystems 3730/3730xl
DNA Analyzers and 3130/3130xl Genetic Analyzers . . . . . . . . . . . . 3
The SNaPshot Multiplex Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
The SNaPshot Multiplex System. . . . . . . . . . . . . . . . . . . . . . . . 3
Preparing the 3730/3730xl and 3130/3130xl Analyzers for
Any5Dye and E5 Dye Set Runs. . . . . . . . . . . . . . . . . . . . . . . . . 4
Creating the Spectral Calibration Protocol . . . . . . . . . . . . . . . . 5
Correcting Spectral Calibration Failures . . . . . . . . . . . . . . . . . . 7
Creating Run Modules. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Creating Regular Run Protocols . . . . . . . . . . . . . . . . . . . . . . . . 9
Rapid Assessment of SNaPshot Primers for Multiplexing
with the SNaPshot Primer Focus Kit . . . . . . . . . . . . . . . . . . . . 11
Data Analysis with GeneMapper Software v3.7 or higher . . . 12
Sizing Differences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Appendix A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
Appendix B . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Appendix C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
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Applied Biosystems 3730/3730xl DNA Analyzers and 3130/3130xl Genetic Analyzers
Overview
The SNaPshot
Multiplex System
with POP-7
Polymer Protocol
This user bulletin provides a detailed protocol for running the SNaPshot® Multiplex
system with POP-7 polymer on the Applied Biosystems 3730/3730xl DNA
Analyzers and the Applied Biosystems 3130/3130xl Genetic Analyzers.
Protocol
Validation and
Testing
Applied Biosystems has not performed optimization or testing to validate or support
the use of the application described in this user bulletin on the 3730/3730xl DNA
analyzers or the 3130/3130xl Genetic analyzers. However, several Applied
Biosystems customers are currently running the SNaPshot Multiplex system and
POP-7 Polymer as described in this protocol. If you want additional information
about these applications, contact Applied Biosystems Technical Support. See
“Appendix A” on page 14 for a table of run configurations using the SNaPshot
Multiplex system on various Applied Biosystems instruments.
Customer
Acknowledgement
We would like to acknowledge Dr. Sylvie Quiniou and Mary Duke at USDA-ARS,
Mid-South Area Genomics Laboratory, Catfish Genetics Research Unit, Stoneville,
MS (http://www.ars.usda.gov) for the protocols and data supplied for this User
Bulletin as well as for the BAC Fingerprinting on 3730/3730xl Analyzers
Application Note (PN 107AP04-01). Dr. Quiniou, a microbiologist at the Catfish
Genetics Research Unit, which focuses on the genetic improvement of catfish in
areas such as growth and disease resistance, has implemented the BAC
fingerprinting application using the SNaPshot reagents-based chemistry system on
the 3730 instrument as part of their research project. We would also like to
acknowledge Mike Zianni and his staff at the Plant Microbe Genomics Facility, Ohio
State University (www.biosci.ohio-state.edu/~pmgf), a core laboratory that
provides DNA sequencing and genotyping services to the university and other
research facilities in the state, for the protocols and data they supplied in this User
Bulletin.
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User Bulletin
SNaPshot Multiplexing on the Applied Biosystems 3730/3730xl DNA Analyzers and 3130/3130xl Genetic Analyzers
SNaPshot Multiplexing on the Applied Biosystems
3730/3730xl DNA Analyzers and 3130/3130xl Genetic
Analyzers
The SNaPshot
Multiplex Kit
The SNaPshot® Multiplex Kit, based on a single nucleotide primer extension
(Figure 1), is part of a versatile system that can be used for a variety of applications:
•
•
•
•
•
Low-to-medium throughput linkage and association studies
Single locus fragment analysis
BAC fingerprinting
Single Nucleotide Polymorphism (SNP) genotyping
Bacterial classification
Figure 1
Single nucleotide primer extension
The SNaPshot reagent-based chemistry is based on the dideoxy single-base
extension of an unlabeled oligonucleotide primer or primers. This method makes for
easy conversion from manual PCR detection methods such as ethidium bromide gelbased chemistries.
The SNaPshot
Multiplex System
The SNaPshot Multiplex system consists of the following:
• SNaPshot® Multiplex Kit including the master mix with fluorescently labeled
ddNTPs and enzymes
• GeneScan™ 120 LIZ® Size Standard
• GeneMapper® Software v3.7 or higher
• Capillary electrophoresis instruments
See “Appendix B” on page 15 for a list of materials and part numbers.
Using the SNaPshot® Multiplex System with the POP-7™ Polymer on Applied Biosystems 3730/3730xl DNA Analyzers and
3130/3130xl Genetic Analyzers
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Preparing the
3730/3730xl and
3130/3130xl
Analyzers for
Any5Dye and E5
Dye Set Runs
Before beginning the setup procedures necessary for running SNaPshot Kit samples
on the 3730/3730xl or 3130/3130xl analyzers with POP-7 polymer, complete the
following steps:
• Install GeneMapper Software v3.7 or higher. Your Applied Biosystems service
engineer or field application specialist can provide instructions for software
installation, if needed.
• Create protocols for the Spectral Calibration and Regular run types with the
Any5Dye dye set for running on 3730/3730xl or with the E5 dye set for running
on 3130/3130xl. (See “Creating the Spectral Calibration Protocol” on page 5.)
• Create a new Run Module with GeneMapper36_POP7 settings for running on
3730/3730xl or with FragmentAnalysis36_POP7 settings for running on
3130/3130xl. (See “Creating Run Modules” on page 8.)
IMPORTANT! The GeneMapper Software must be installed on the instrument
computer.
Additional information can be found in the Applied Biosystems 3730/3730xl DNA
Analyzers User Guide for use with Data Collection Software v2.0 (PN 4347118) and
the Applied Biosystems 3130/3130/xl Genetic Analyzers Getting Started Guide
(PN 4352715).
Both documents can be ordered from the Applied Biosystems Web site at
www.appliedbiosystems.com.
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User Bulletin
SNaPshot Multiplexing on the Applied Biosystems 3730/3730xl DNA Analyzers and 3130/3130xl Genetic Analyzers
Creating the
Spectral
Calibration
Protocol
Create the spectral calibration protocol before you process the run. This protocol
uses the Any5Dye dye set with the 3730/3730xl analyzer, which is available only
with Data Collection Software v2.0 or higher, or the E5 dye set with the 3130/3130xl
analyzer, which is available with Data Collection Software v3.0.
To create a spectral calibration protocol:
1. In the left-hand navigation window of the Data Collection software, select
Protocol Manager.
2. In the Instrument Protocols area, select New to open the Protocol Editor
window.
Dye Set setting for
the 3730/3730xl
DNA Analyzer
Run Module setting for the
3730/3730xl DNA Analyzer
wind
Figure 2 A spectral protocol in Protocol Manager, using the Any5Dye dye set
and the Spect36_MtxStd_POP7_1 run module on the 3730/3730xl DNA Analyzer
Dye Set setting for
the 3130/3130xl
Genetic Analyzer
Run Module setting for
the 3130/3130xl
Genetic Analyzer
Figure 3 A spectral protocol in Protocol Manager, using the E5 dye set and the
Spect36_POP7_1 run module on the 3130/3130xl Genetic Analyzer
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3. Enter a name for your spectral calibration in the Name field (E5_Spectral in the
example shown in Figure 3 on page 5). Use Table 1 below to make the
appropriate selection for your instrument from each field listed.
Note: Entering information in the Description field is optional
Table 1 Protocol Editor selections for the 3730/3730xl and the 3130/3130xl
analyzers
Field
3730/3730xl Setting
3130/3130xl Setting
Type
Spectral
Spectral
Dye Set
Any5Dye
E5
Polymer
POP7
POP7
Array Length
36
36
Chemistry
Matrix Standard
Matrix Standard
Run Module
Spect36_MtxStd_POP7_1
Spect36_POP7_1
4. Select Edit Parameter at the bottom of the Protocol Editor window to open the
Edit Spectral Params window.
Figure 4
The Edit Spectral Params window
– For the 3730/3730xl DNA analyzers, the Matrix Condition Number Bound
can be set to 2.5 (minimum) and 4.5 (maximum). The Minimum Quality
Score should not be lower than 0.9. A minimum quality score of 0.95 is
common.
– For the 3130/3130xl Genetic analyzers, use the default condition numbers.
5. Click OK to save any changes to the parameters and click OK again to save the
newly created Spectral Protocol.
6. Prepare a spectral calibration matrix plate by following the protocol provided
with the Matrix Standard Set DS-02 Kit (PN 4323014 for the 3130 series
systems and PN 4322849 for the 3730 series systems). PN 4322849 is
formulated for use with the ABI PRISM® 3700 DNA Analyzer, but it can also
be used on the 3730/3730xl instrument.
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User Bulletin
SNaPshot Multiplexing on the Applied Biosystems 3730/3730xl DNA Analyzers and 3130/3130xl Genetic Analyzers
7. Follow instructions in the instrument user manual to perform a spectral
calibration run. When the spectral calibration run is complete and passes (>90%
of passing caps), the instrument is ready to run SNaPshot samples. (See
“Creating Regular Run Protocols” on page 9.)
If the spectral calibration fails, check the Spectral Calibration Run Log for the
error source. Any errors with spectral calibration in each capillary are also
detailed in the log file. The file is located at the following address:
E://AppliedBiosystems/UDC/DataCollection/Data/ga3x30/Instrument
Number/SpectralCalTmpFiles/Any5Dye/SpectralCalResult-Run
Correcting
Spectral
Calibration
Failures
If the spectral calibration failure is due to matrix standard peaks being too strong
(i.e., if pull-up or pull-down peaks are visible (Figure 5), dilute the standards 2:1 with
Hi-Di™ Formamide and perform spectral calibration again.
CHEMICAL HAZARD. Hi-Di Formamide. Exposure causes
eye, skin, and respiratory tract irritation. It is a possible developmental and birth
defect hazard. Read the MSDS, and follow the handling instructions. Wear
appropriate protective eyewear, clothing, and gloves.
Figure 5
Failed spectral calibration showing pull-up and pull-down peaks
Using the SNaPshot® Multiplex System with the POP-7™ Polymer on Applied Biosystems 3730/3730xl DNA Analyzers and
3130/3130xl Genetic Analyzers
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If the spectral calibration failure is due to the condition number not being within the
designated range, adjust the Matrix Condition Number Bound ‡ by following the
steps below.
Note: The capillary condition number for each capillary is found in the Spectral
Calibration Run Log.
1. Determine the average condition number for the array by selecting several wells.
2. Adjust the matrix condition number bounds to approximately ±1.5 of the
average condition number.
3. Run spectral calibration immediately after you correct the reason for failure.
Make new matrix standards or, if the elapsed time between the two spectral runs
is less than one hour, use the same matrix standards.
Creating Run
Modules
To create a Run Module:
1. Launch the Data Collection Software v3.0 for the 3130 Series Systems or v2.0
or higher for the 3730 Series Systems.
2. Select Module Manager and then New to open the Run Module Editor window.
Template setting for the
3730/3730xl DNA analyzer
Figure 6 The Run Module Editor window using the GeneMapper36_POP7
template on the 3730/3730xl DNA analyzer
‡ This parameter is adjusted only if the spectral calibration failed and the condition number is
not within the designated range. The capillary condition number is located in the Spectral
Viewer.
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SNaPshot Multiplexing on the Applied Biosystems 3730/3730xl DNA Analyzers and 3130/3130xl Genetic Analyzers
Template setting for the
3130/3130xl Genetic analyzers
Figure 7 The Run Module Editor window using the FragmentAnalysis36_POP7
template on the 3130/3130xl Genetic analyzer
3. Fill in the appropriate fields in the Run Module setting fields, as follows:
Field
3730/3730xl Instrument Setting
3130/3130xl Instrument Setting
Name
E5 Chemistry (for example)
E5 Chemistry (for example)
Type
Regular
Regular
Template
GeneMapper36_POP7
FragmentAnalysis36_POP7
Note: For SNaPshot reagent-based chemistry, if the desired fragments appear before
the default time of 1,200 seconds, the Run_Time may be shortened. The Plant
Microbe Genomics Facility at Ohio State University shortened the Run_Time to 600
seconds on a 36-cm capillary array.
Creating Regular
Run Protocols
To create Regular Run Protocols, follow theses steps:
1. Open the Data Collection software and select Protocol Manager.
2. Click the New button in the Instrument Protocols area. The Protocol Editor
window opens (see Figure 8 and Figure 9 on page 10).
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Dye Set setting for
the 3730/3730xl
DNA Analyzer
Figure 8 The Protocol Editor window in the Data Collection software on the
3730/3730xl DNA analyzer
Dye Set setting for
the 3130/3130xl
Genetic Analyzer
Figure 9 The Protocol Editor window in the Data Collection software on the
3130/3130xl Genetic analyzer
3. Enter the name of your new instrument protocol (i.e., E5_Chemistry) in the
Name field. Entering information in the Description field is optional.
4. Select Regular in the Type field.
5. In the Run Module field, select the Run Module created above
(i.e. E5_Chemistry) from the drop-down menu.
6. In the Dye Set field, select Any5Dye for running on the 3730/3730xl analyzer or
E5 for running on the 3130/3130xl. Then click OK.
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SNaPshot Multiplexing on the Applied Biosystems 3730/3730xl DNA Analyzers and 3130/3130xl Genetic Analyzers
7. Set up a sample sheet. Refer to instructions in the Applied Biosystems
3130/3130xl Genetic Analyzers Getting Started Guide (PN 4352715) or the
Applied Biosystems 3730/3730xl DNA Analyzer Getting Started Guide
(PN 4359476).
8. Run the sample plates and analyze the results with GeneMapper Software v3.7
or higher for simultaneous sizing and genotyping (see Figure 10).
Figure 10 shows a multiplexed SNaPshot Kit reaction using POP-7 polymer and a
36-cm capillary array on the Applied Biosystems 3730 DNA analyzer. The data was
analyzed by GeneMapper Software v3.7. The color bars represent bins used for allele
calling. The GS120LIZ size standard was used for sizing.The analyzed data can then
be exported in a tab-delimited Microsoft Excel® Software spreadsheet.
Figure 10 Multiplexed SNaPshot Kit reaction using POP-7 polymer and a 36-cm
array on the 3730 DNA analyzer
Rapid
Assessment of
SNaPshot
Primers for
Multiplexing with
the SNaPshot
Primer Focus Kit
During the course of SNP validation, users often encounter two major obstacles:
• Determining which combination of SNP loci to multiplex
• Establishing tools to enable automatic data analysis
Using the SNaPshot Primer Focus kit enables rapid assessment of potential
oligonucleotides used in the single-base extention step designed for a project. The
chemistry uses single-base extension of a primer in the presence of fluorescentlylabeled dideoxynucleotides, but without the presence of DNA template. You can
preview all potential full length products (the four possible alleles are represented by
four colors: A=green, C=black, G=blue, T=red), and you can calculate the mobility
rate for each allele using this kit.
Once the data is generated, the optimal combination of SNP markers are identified
for multiplexing. In Figure 11 and Figure 12 on page 12, Focus kit data from a
3130xl Genetic Analyzer demonstrates the mobility of a 32-mer oligonucleotide by
itself and in combination with a 36-mer oligonucleotide. The data shows that
sufficient separation is obtained from the 4-nucleotide length difference between the
two primers.
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dR6G
dR110
dTAMRA™
dROX™
Figure 11 Primer Focus® Kit samples run with POP-7 polymer on a 3130xl
Genetic Analyzer with a 36-cm capillary array. Mobility of a 32-mer
oligonucleotide primer focus product is shown for each of the 4 dyes: dR110
(blue), dR6G (green), dTAMRA™ (yellow) and dROX™ (red).
Figure 12 Multiplexed Primer Focus Kit samples run with POP-7 polymer on a
3130xl Genetic Analyzer with a 36-cm capillary array. Mobility of two primer focus
oligonucleotide products, the 32-mer shown in Figure 11 and a 36-mer when
mixed in equal amounts and run in the same capillary.
Data Analysis
with GeneMapper
Software v3.7 or
higher
The workflow for analysis of data in GeneMapper software involves the following:
•
•
•
•
Addition of appropriate samples to a new project
Setting up analysis parameters
Running the analysis
Reviewing the results of the analyzed project
The Autoanalysis Option in the Data Collection software run on the 3130/3130xl
Genetic Analyzers and the 3730/3730xl DNA Analyzers automates all steps from
addition of samples to running the analysis; the user only reviews the analyzed
results. GeneMapper software contains a SNaPshot Kit specific analysis method
designed to recognize and analyze SNaPshot Kit data. Once the mobility of the
extended products has been determined, bins representing the actual allele size range
and dye color for a given SNP can be assigned. The software uses bins to make the
actual allele call (see Figure 10 on page 11). Once these bins have been set up, the
software automatically assigns genotypes to samples at the end of the analysis.
These bins are saved and reapplied automatically to all subsequent projects that use
the same set of SNPs enabling a streamlined workflow from sample loading, data
collection, data analysis and genotype results generation. Please refer to the
GeneMapper® Software v3.7 User Guide (PN 4359413) for more detailed
information on data analysis.
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Sizing
Differences
In fragment analysis, a fragment is assigned a size based on its relative mobility to
size standards as it migrates through the polymer. Any change in run conditions,
such as capillary array length or polymer type, affects fragment mobility. Sizing
differences resulting from mobility changes between various types of polymer are
more apparent for sequences < 50 base pairs (bp). Due to the nature of the polymer,
smaller fragments (< 50 bp) run on POP-7™ polymer on the 3130/xl Genetic
Analyzers and on the 3730/xl DNA Analyzers may have slightly different mobilities.
To avoid sizing inconsistency with smaller fragments, Applied Biosystems
recommends that genotyping projects be started and completed on the same
instrument, using consistent run conditions. Avoid inconsistencies by creating
single-base extention primers no shorter than 25 nucleotides (nts). In addition, the
spacing should be at least 6-8 nts for primers that are less than 30 nts and 4-6 nts for
primers that are greater than 30 nts. Alternatively, genotypes can be standardized to
one platform by running representative alleles across all platforms and creating
instrument-specific bin sets with standardized bin names in GeneMapper Software
v3.7 or higher.
Conclusion
In addition to multiplexing, the SNaPshot Multiplex Kit can also be used for the
rapidly expanding field of fragment analysis and SNP genotyping. Researchers in
these areas increasingly look for ways to improve throughput and cost-effectiveness
without sacrificing data quality. One solution is to leverage the flexibility of the
Applied Biosystems 3730/3730xl DNA analyzers and the 3130/3130xl Genetic
analyzers to accommodate genotyping applications designed for the SNaPshot
Multiplex Kit.
For more information, please contact your Sales Representative, Field Application
Specialist, or Technical Support.
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Appendix A
Throughput Results Using the SNaPshot Multiplex System on the Applied
Biosystems 3730/3730xl DNA Analyzers, 3130/3130xl Genetic Analyzers,
and 3100 Analyzers
Instrument
Number of
Capillaries
Run Time
(min)
Number of
Runs/24 hr
Number of
Genotypes/Run
Number of
Genotypes/Day
3730
(36-cm, POP-7)
48
33
43
10
20,640
3730xl
(36-cm POP-7)
96
33
43
10
41,280
3130
(36-cm, POP-7)
4
35
41
10
1,640
3130
(22-cm, POP-4™)
4
20
72
10
2,880
3100-Avant
(36-cm, POP-4)
4
24
60
10
2,400
3100-Avant
(22-cm POP-4)
4
20
72
10
2,880
3130xl
(36cm, POP-7)
16
35
41
10
6,560
3130xl
(22-cm POP-4)
16
20
72
10
11,520
3100
(36-cm array POP-4)
16
24
60
10
9,600
3100
(22-cm array POP-4)
16
20
72
10
11,520
(array & polymer)
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Appendix B
Appendix B
Ordering Information for Materials
Description
Comments
PN
ABI PRISM® SNaPshot® Multiplex Kit
5,000 rxns
4323163
ABI PRISM® SNaPshot® Multiplex Kit with protocol
1,000 rxns
4323154
ABI PRISM® SNaPshot® Multiplex Kit with protocol
100 rxns
4323151
SNaPshot® Primer Focus® Kit
100 rxns
4329538
3730/3730xl DNA Analyzer User’s Guide v2.0 or higher
4347118
Applied Biosystems 3130/3130xl Genetic Analyzers Getting Started Guide
4352715
GeneMapper® Software v3.7
Initial License
4363138
GeneMapper® Software v3.7
Additional License
4363137
GeneScan™ 120 LIZ® Size Standard
4324287
48 capillary × 36 cm Capillary Array (3730)
4331247
96 capillary × 36 cm Capillary Array (3730xl)
4331244
36cm Applied Biosystems 3130 and 3100-Avant Capillary Array
4333464
36cm Applied Biosystems 3130xl and 3100 Capillary Array
4315931
Hi-Di™ Formamide
4311320
Matrix Standard Set DS-02 (dR110, dR6G, dTAMRA™, dROX™, LIZ® Dyes)
3700 DNA Analyzer
4322849
Matrix Standard Set DS-02 (dR110, dR6G, dTAMRA™, dROX™, LIZ® Dyes)
3130/3130xl Genetic
Analyzers
4323014
POP-7™ Polymer for Applied Biosystems 3130 and 3130xl Genetic
Analyzers
7 mL
4352759
POP-7™ Polymer for 3730/3730xl DNA Analyzers, Box of 15, 25 mL
Bottles
15 bottles/box 420 mL
4335612
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3130/3130xl Genetic Analyzers
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Appendix C
SNaPshot Reference Documents
Title
Part Number
ABI PRISM® SNaPshot™ ddNTP Primer Extension Kit Protocol
4312665
ABI PRISM® SNaPshot™ Multiplex Kit Protocol
4323357
ABI PRISM® SNaPshot™ Multiplex Kit Quick Reference Card
4323975
BAC Fingerprinting on the Applied Biosystems 3730/3730xl DNA Analyzer
SNaPshot® Multiplex System Application Note
107AP04-01
GeneMapper® Software Version 4.0 SNaPshot Kit Analysis Getting Started Guide
4363078
GeneScan Analysis Software: Genetic Analysis: Product Bulletin
107PB05-01
Using ABI PRISM® Genotyper® Software Version 3.7 with the SNaPshot™ Multiplex
Kits ABI PRISM® Genotyper® Software User Bulletin
4326549
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User Bulletin
© Copyright 2005, 2010 Applied Biosystems. All rights reserved.
For Research Use Only. Not for use in diagnostic procedures.
Information in this document is subject to change without notice. Applied Biosystems assumes no responsibility for any errors that may appear in this document.
This document is believed to be complete and accurate at the time of publication. In no event shall Applied Biosystems be liable for incidental, special, multiple, or
consequential damages in connection with or arising from the use of this document.
SNaPshot Kits For Research Use Only. Not for use in diagnostic procedures.
NOTICE TO PURCHASER: LIMITED LICENSE
The purchase price of the ABI PRISM SNaPshot Multiplex Kit includes a limited, nontransferable license under U.S. patents or their foreign counterparts,
to use only this amount of the product for DNA Sequencing and related processes described in said patent solely for the research and development activities of
the purchaser. No license under these patents to use the PCR Process is conveyed expressly or by implication to the purchaser by the purchase of this product.
A license to use the PCR Process for certain research and development activities accompanies the purchase of certain reagents from licensed suppliers such as
Applied Biosystems when used in conjunction with an Authorized Thermal Cycler, or is available from Applied Biosystems. Further information on purchasing
licenses to practice the PCR Process may be obtained by contacting the Director of Licensing at Applied Biosystems, 850 Lincoln Centre Drive, Foster City,
California 94404, or at Roche Molecular Systems, Inc., 1145 Atlantic Avenue, Alameda, California 94501.
NOTICE TO PURCHASER: LIMITED LICENSE
This kit (reagent) is sold pursuant to a limited sublicense from Amersham International plc under one or more U.S. Patent Nos. 5,498,523, 5,614,365, and
corresponding foreign patents and patent applications. The purchase of this kit (reagent) includes a limited non-exclusive sublicense (without the right to resell,
repackage, or further sublicense) under such patent rights to use this reagent for DNA sequencing or fragment length analysis solely with an Applied Biosystems
commercial automated sequencing machine or other authorized DNA sequencing machines that have been authorized for such use by Applied Biosystems, or
for manual DNA sequencing. No license is hereby granted for the use of this kit, or the reagents therein in any other automated sequencing machine. Such
sublicense is granted solely for research and other uses that are not unlawful. No other license is granted expressly, impliedly, or by estoppel. For information
concerning the availability of additional license to practice the patented methodologies, contact: Amersham Life Science, Inc., Vice President, Regulatory Affairs,
P.O. Box 22400, Cleveland, Ohio 44122. Patents are pending in countries outside the United States.
End User License
The purchase price of this product includes a limited, non-transferable license under U.S. Patent Nos. 5,888,819, 6,004,744 and their foreign counterparts owned
by Orchid Biocomputer, Inc. of Princeton, New Jersey, to perform 24, 100, or 1000 Genotypes (For purposes of this End User License, Genotype means the detection or quantification of an individual SNP within a single sample.) solely for the detection and analysis of SNPs in samples for research purposes only and only on
automated electrophoresis nucleotide sequencers. This license specifically excludes performing services for a third party and any and all medical diagnostic or
therapeutic uses. Information about purchasing licenses to practice primer extension technology covered by Orchid BioSciences, Inc. patents for any other use
may be obtained by contacting the Senior Director for Business Development at Orchid BioSciences, Inc. Princeton, New Jersey, U.S.A. at (609) 750-2200.
3730/3730xl and 3130/3130xl Analyzers NOTICE TO PURCHASER:
This instrument is Authorized for use in DNA sequencing and fragment analysis. This authorization is included in the purchase price of this instrument and
corresponds to the up-front fee component of a license under process claims of U.S. patents and under all process claims for DNA sequence and fragment
analysis of U.S. patents now or hereafter owned or licensable by Applied Biosystems for which an Authorization is required, and under corresponding process
claims in foreign counterparts of the foregoing for which an Authorization is required. The running royalty component of licenses may be purchased from Applied
Biosystems or obtained by using Authorized reagents purchased from Authorized suppliers in accordance with the label rights accompanying such reagents.
Purchase of this instrument does not itself convey to the purchaser a complete license or right to perform the above processes. This instrument is also licensed
under U.S. patents and apparatus and system claims in foreign counterparts thereof. No rights are granted expressly, by implication or by estoppel under
composition claims or under other process or system claims owned or licensable by Applied Biosystems. For more information regarding licenses, please
contact the Director of Licensing at Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404, USA.
NOTICE TO PURCHASER:
The purchase price of the Applied Biosystems 3730 and 3730xl DNA Analyzers and 3130 and 3130xl Genetic Analyzers includes a grant of a limited, nontransferable license under U.S. patents and method claims of its foreign counterparts, and element claims of its foreign counterparts, to use this particular instrument
for electrophoresis methods employing fluorescence as a means of detection. No other licenses or rights are hereby conveyed either expressly, by implication, or
estoppel including, but not limited to, any claims to a composition.
The Applied Biosystems 3730 and 3730xl DNA Analyzers and 3130 and 3130xl Genetic Analyzers s include patented technology licensed from Hitachi, Ltd. as part
of a strategic partnership between Applied Biosystems and Hitachi, Ltd., as well as patented technology of Applied Biosystems.
TRADEMARKS:
Applied Biosystems, ABI PRISM, GeneMapper, LIZ, and SNaPshot are registered trademarks and AB (Design), Applera, GeneScan, Hi-Di, POP-4, POP-7, ROX, and
TAMRA are trademarks of Applied Biosystems or its subsidiaries in the U.S. and/or certain other countries.
All other trademarks are the sole property of their respective owners.
19
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July 19, 2005 9:15 am, Multiplex SNaPshot UB.fm
User Bulletin
Refer to page 19 for Licensing and Trademark information.
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06/2010
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Part Number 4367258 Rev. B